Various methods of breaking Abbe's diffraction limit in microscopy have been developed in the state of the art. The term “high resolution” used in this specification addresses a resolution beyond Abbe's diffraction limit. A method abbreviated to PALM (photo activated light microscopy) is known from WO 2006/127692 and DE 102006021317 A1, which uses labels for imaging a sample which labels can be activated e.g. by means of optical radiation. WO 2006/127692 and DE 102006021317 A1, and respective counterparts U.S. Pat. No. 7,626,703 B2 and U.S Patent Publication No. 2011/0160083 A1 are hereby fully incorporated herein by reference. The labels can emit specific fluorescence radiation only in the activated state. Non-activated labels emit no, or at least no noticeable, fluorescence radiation even after irradiation by excitation radiation. The activation radiation is therefore generally called a switching signal. In the PALM method, the switching signal is applied such that at least a certain proportion of activated labels are spaced apart from neighbouring activated labels such that these labels are separated, as measured by the optical resolution of the microscopy, or can be separated subsequently by image processing methods. It is said that at least some of the fluorescence labels are isolated. After recording the fluorescence radiation, for these isolated emitters the centre of their diffraction-limited images is identified. From this, the position of the labels in the sample can be determined computationally with higher precision than the optical resolution itself allows. This procedure is called localization. The increased resolution by computational determination of the centre of the diffraction-limited images achieves a so called “superresolution”. It requires at least some of the activated labels to be distinguishable with the optical resolution, i.e. these activated labels are isolated in the sample. Then their position can be determined with higher precision and they are localized.
To isolate individual labels, the PALM principle utilizes statistical effects. In the case of a label which can be excited to fluorescence radiation only after receiving the switching signal of given intensity, one can ensure, by adjusting the intensity of the switching signal, that the probability of activating labels present in a given unit area of the sample is so small that there are enough domains in which only labels emit fluorescence radiation which are distinguishable with the optical resolution.
The PALM principle was refined with respect to the activation of the labels to be recorded. Thus for example, in the case of labels that have a long-lived non-fluorescing and a short-lived fluorescing state, a separate activation with activation radiation differing spectrally from the excitation radiation is not necessary. Instead the labels of or in the sample are first activated with high-intensity illumination radiation such that the vast majority of the labels is converted to the non-fluorescence-capable long-lived state (e.g. a triplet state). The remaining, still fluorescing labels are thereby isolated in terms of the optical resolution.
It may also be noted that the PALM principle has meanwhile also acquired other abbreviations, such as for example STORM, in the technical literature. In this description, the abbreviation PALM is used for all microscopy techniques which achieve a spatial resolution beyond the optical resolution of the apparatus used by first isolating and then localizing fluorescent molecules. The PALM method has the advantage that no particular high spatial resolution is required for illumination. A simple widefield illumination suffices for activation and/or excitation.
The PALM method uses a sample comprising fluorescent substances. The fluorescent molecules (either all or at least a sub-set of all molecules) are first isolated in terms of the optical resolution of the imaging of the sample and then localized to determine the localization of the molecules with a resolution beyond the diffraction limit of the optical imaging. This methodology can be worked with as soon as the sample has certain fluorescence properties, as explained above. These properties can be achieved by providing the sample with staining material or dies etc. Biological samples can be provided with respective fluorescence material by e.g. transfection. Of course, it is also possible to use a sample which already comes with the respective fluorescence molecules without need to prepare the sample. The term “label” used in this specification shall cover all these options, in particular it shall cover embodiments where a sample preparation step is performed to provide the sample with respective fluorescent molecules and embodiments where the sample already comprises such fluorescent molecules.
The PALM principle requires many frames of the sample to be recorded, each of which contains labels being isolated. In order to image the sample completely, all frames together should ensure that if possible all labels were at least once isolated. As each frame contains a sub-set of the labels—some of these (but not necessarily all) are isolated, the PALM method usually requires a large number of frames, which means that it takes a certain period of time to record a complete image. This is associated with a considerable computational outlay as a large number of labels must be computationally localized in each frame. Large amounts of data are accumulated.
There is now a need not only to record high-resolution images in a colour channel, but to obtain colour information, i.e. an indication of the wavelength of the fluorescing emitters. In fluorescence microscopy, the state of the art knows a microscope according to FIG. 8. The fluorescence microscope 100 shown there comprises an illumination beam path 3 as well as an imaging beam path 4 which illuminate a sample 2 with excitation radiation via a common objective 5 and image the fluorescing sample 2. The illumination beam path 3 is combined with the imaging beam path 4 via a beam splitter 6, usually dichroic in design, such that both illumination radiation from the illumination beam path 3 is incident on the sample 2 through the objective 5 and the imaging of the sample is carried out through the objective 5 and via the imaging beam path 4. The illumination beam path 3 usually has several spectral channels; in the representation in FIG. 8 two laser sources L1 and L2 are shown by way of example, the radiation of which is merged via a beam splitter 5. The illumination beam path 3 thus illuminates the sample 2 with radiation of at least two wavelengths, with the result that a multicolour fluorescence excitation of the sample 2 is effected. The sample 2 also emits multicoloured fluorescence radiation (naturally this could also be the case for a monochrome fluorescence excitation and different fluorescence molecules which fluoresce at different wavelengths). In the imaging beam path 4, the image of the sample 2 is therefore divided into e.g. three colour channels, i.e. directed to three cameras K1, K2 and K3, via two beam splitters 7 and 8 as well as suitable lens systems not described in more detail. The split by the beam splitters 8 and 9 effects a spectrally selective division towards the cameras K1 to K3. Alternatively or in addition, suitable colour filters can also be used. Several colour channels are thus obtained, one for each camera. However, a disadvantage of this design is that the camera systems used are very expensive due to the high diffraction-limited resolution required. Furthermore, the installation space for the microscope 100 is large on account of the required beam paths and colour splitters. Usually, the cameras must be cooled and likewise require a large installation space. A further problem is that the cameras K1, K2 and K3 must be aligned precisely relative to one another so that the images of the individual colour channels are aligned correctly relative to one another. Any alignment error between the beam paths of the individual colour channels would result in a colour aberration generating a chromatic aberration in the image of the sample.
One could use the microscope 100 of FIG. 8 for the PALM principle, but then the amount of data accumulating as well as the computational outlay would be multiplied by the number of colour channels.